Acute Shock Induced by Antigen Vaccination in NOD Mice
1 Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgiumh%tj, 百拇医药
2 Clinic of Endocrinology, University Hospital "St. Spiridon," University of Medicine and Pharmacy "Gr. T. Popa," Iasi, Romaniah%tj, 百拇医药
3 Laboratory for Immunology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgiumh%tj, 百拇医药
ABSTRACTh%tj, 百拇医药
Type 1 diabetes in NOD mice can be prevented through autoantigen vaccination by shifting lymphocyte differentiation toward a T-helper 2 (Th2) response. However, in other models of autoimmunity, this approach may be accompanied by unexpected triggering of Th2-dependent anaphylactic shock. To test the safety of vaccination therapy in the NOD mouse model, we evaluated the effects of immunization with a wide battery of antigens in NOD, BALB/c, and C57BL/6 mice. Surprisingly, a nondiabetogenic antigen, hen egg white lysozyme, induced severe shock exclusively in NOD mice (shock in 11 of 11 mice, lethal in 3 mice). Shock severity was further increased by a more pronounced Th2 setting generated by 1{alpha} ,25(OH)2D3 administration (17 of 17 mice, lethal in 14 mice, P < 0.0001). Pretreatment with dexamethasone resulted in full rescue, indicating an immune-mediated mechanism. Serum IgE levels and Th1/Th2 cytokine profile analysis showed that the shock phenomenon was paralleled by a Th2 response. mRNA expression of platelet-activating factor receptor (PAF-R) was significantly higher in NOD mice (P < 0.01) and was further increased by 1{alpha} ,25(OH)2D3. Pretreatment with WEB2086 (PAF-R antagonist) again protected all mice from lethal shock, indicating PAF as an anaphylaxis effector. In conclusion, in NOD mice, vaccination leading to a Th2 immune shift can result in a lethal anaphylactic reaction.
Type 1 diabetes is caused by immune destruction of insulin-producing ß-cells in the pancreatic islets of Langerhans (1). In the NOD mouse, a widely used animal model for the study of type 1 diabetes, lymphocytic infiltration into the pancreatic islets (insulitis) is the primary lesion that eventually progresses to overt diabetes. Evidence is accumulating that type 1 diabetes is mediated by T-helper 1 (Th1) cells and that protection or prevention of the disease can be achieved by shifting the immune response toward Th2 activation (2,3).qc'r, 百拇医药
Several diabetogenic autoantigens have been described, such as GAD, insulin, and heat shock protein, in both animal models and human disease (4,5). Furthermore, epitope spreading and molecular mimicry is enlarging their spectrum during disease evolution (5). The biochemical properties of antigens together with the genetic background of the immune system significantly influence the type (Th1 or Th2) of immune activation (6). Many studies have investigated the effects of administration of diabetes relevant autoantigens or their immunodominant peptides in the NOD mouse model. Most studies convincingly demonstrate a prevention or delayed onset of disease, associated with a shift toward a more Th2-biased response (5,7). Recently, also in type 1 diabetic patients, peptide treatment with specific autoantigens has been shown to prevent or delay the disease and was concluded to be safe (8).
This vaccination-triggered shift from autoreactive Th1 toward Th2 cells has not only been observed in NOD mice (9) but also in experimental autoimmune encephalomyelitis (EAE), another Th1-mediated autoimmune disease (10). Nevertheless, the administration of self-antigen in the EAE model has lead to unforeseen consequences of Th2 activation. Indeed, vaccination with the myelin proteolipid peptide PLPp(139–151) in SJL/J mice under certain conditions, essentially depending on the time point of peptide rechallenge, unexpectedly resulted in lethal anaphylaxis (11). Also in the human situation, a recent phase II trial in which multiple sclerosis (MS) patients received an altered peptide ligand from a myelin basic protein epitope, repeated injections with the peptide induced immediate hypersensitivity reactions in 9% of the subjects (12). These reports suggest that an autovaccine-triggered immune response may produce extremely undesirable allergic reactions. To what extent anaphylaxis depends on the genetic background, the antigen type, or the timing of vaccination is not yet completely known.
The aim of the present study was to test whether vaccination in NOD mice, animals having an immune system genetically prone to destroy pancreatic islets through Th1-mediated mechanisms, is perfectly safe or whether life-threatening reactions can be induced by manipulating the immune response. Indeed, the NOD mouse strain also displays immune system particularities beyond Th1-dependent autoimmune islet destruction because they seem to be highly susceptible to anaphylactic shock under certain specified conditions (13,14). NOD mice might therefore behave differently from other mouse strains in certain situations of immune challenge (13). To investigate whether vaccination could induce lethal shock in NOD mice, we tested a wide panel of antigens. We compared the effects of several diabetes-relevant autoantigens and diabetes-irrelevant peptides or proteins in NOD mice to the effects obtained in two other mouse strains (BALB/c and C57BL/6 mice), in which a Th1-activated immune system is absent. Based on our previous findings, we also treated mice from various strains with an immunomodulatory agent known to favor an autoantigen-specific Th2 shift in NOD mice, namely 1,25(OH)2D3, and examined its effect on the safety profile of vaccination.
RESEARCH DESIGN AND METHODS1}afv{}, http://www.100md.com
Animals.1}afv{}, http://www.100md.com
NOD mice were originally obtained from Prof. C.Y. Wu in 1989 and further bred in our animal house (Proefdierencentrum, Leuven, Belgium), kept under semi-barrier conditions (15). At the time of the study, diabetes incidence by the age of 200 days in our stock colony was 72% in female mice and 25% in male mice. Nondiabetic mice aged 7–9 weeks of both sexes were used in the experiments. BALB/c and C57BL/6 mice were purchased from Harlan (Horst, the Netherlands). NOD/LtSz-scid/scid (NOD-scid) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and kept under specific pathogen-free conditions. Mice were fed regular diet and water ad libitum. Animals receiving 1{alpha} ,25(OH)2D3 or vehicle were fed a low-calcium vitamin D–replete diet (0.2% calcium, 1% phosphate, 2,000 units vitamin D/kg; Hope Farms, Woerden, the Netherlands).1}afv{}, http://www.100md.com
Proteins and peptides.
Hen egg white lysozyme (HEL) (94% purity), ovalbumin (OVA) (Grade V, 98% purity), keyhole limpet hemocyanine (KLH), bovine insulin B (ins-B) chain, and PLP peptide (amino acids 135–151) were purchased from Sigma (Bornem, Belgium). GAD65 (p524–542) (SRLSKVAPVIKARMMEYGTT), heat shock protein (hsp)-65 peptide (PALDSLTPANED), and tetanus toxin (TT) peptide (p830–843) (QYIKANSKIGITE) contained a COOH-terminal amidation (CONH2) group and were >75% pure (Eurogentec, Luik, Belgium).|5, 百拇医药
Vaccination and intervention regimens.|5, 百拇医药
The 8-week-old NOD, BALB/c, and C57BL/6 mice were immunized by injection of 100 µg antigen (HEL, GAD65, ins-B, hsp65, PLP, OVA, KLH, and TT) emulsified at a 1:1 concentration in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (Difco Laboratories, Detroit, MI) in the hind footpads. NOD-scid mice were injected with 100 µg HEL antigen suspended in CFA. All mice were reinjected with the same antigen 3 weeks later in a similar manner.
NOD, BALB/c, and C57BL/6 mice were treated with 1{alpha} ,25(OH)2D3 from 3 weeks of age until the end of the experiment. The drug was suspended in arachis oil and administered at a dose of 5 µg/kg i.p. every 2 days. Control NOD, BALB/c, and C57BL/6 mice received the treatment vehicle.6., 百拇医药
Dexamethasone (50 µg dissolved in 0.9% NaCl; Aacidexam, NV Organon, Oss, the Netherlands) was injected intraperitoneally in NOD mice 48, 24, and 2 h before the booster injection. WEB2086 (platelet-activating factor receptor [PAF-R] antagonist; Boehringer Mannheim, Mannheim, Germany; a gift from Prof. Vermylen, University of Leuven, Leuven, Belgium), 500 µg dissolved in PBS by sonication, was injected intraperitoneally in NOD mice 10 min before the HEL booster injection.6., 百拇医药
Follow-up of mice.6., 百拇医药
Clinical evolution and survival rate after sensitization with various peptides were monitored in different mouse strains. Shock was characterized by pilo-erection; prostration; erythema of the tail, ears, and footpads; and dyspnea with shallow breathing. Serum for antibody measurement and spleens for quantification of mRNA levels were collected before immunization and again before booster administration.
Measurement of antibody production.{1(!(kz, 百拇医药
HEL-specific IgG1, IgG2a, and IgE antibodies were measured in mouse sera by enzyme-linked immunosorbent assay (ELISA). Briefly, for IgG1 and IgG2a ELISA, 96-well microtiter plates were coated overnight with HEL (10 µg/ml). Samples for IgG1 and IgG2a measurement were diluted at 1:3,000 and 1:800, respectively. Bound IgG1 and IgG2a were detected using biotinylated anti-mouse IgG1 or anti-mouse IgG2a, respectively. The plates were further processed with horseradish peroxidase–conjugated streptavidin and tetramethylbenzidine as substrate. To measure HEL-specific IgE antibody, plates were coated with anti-mouse IgE antibody. After incubation with diluted mouse sera (1:20), bound IgE was incubated with HEL. The plates were then processed with peroxidase-conjugated anti-HEL antibody, and tetramethylbenzidine was used as a substrate. Plates were read at 450 nm on a MultiSkan ELISA reader (ThermoLab Systems, Brussels, Belgium).
Total IgE was measured by sandwich ELISA (BD Biosciences, Erembodegem, Belgium) according to the manufacturer’s instructions. Serum samples were analyzed in duplicate at a 1:10 dilution.]\j!s9|, 百拇医药
Real-time RT-PCR for cytokines, PAF-acetylhydrolase, and PAF-R.]\j!s9|, 百拇医药
Total RNA from spleen was extracted using the SV total RNA isolation system (Promega, Madison, WI). A 1-µg target RNA was reverse-transcribed as described previously (16).]\j!s9|, 百拇医药
Real-time quantitative RT-PCR was performed for ß-actin, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-, IL-12, -interferon (IFN-), IL-4, IL-13, PAF-acetylhydrolase (AH), and PAF-R as previously described (16). The primers and fluorogenic probe for PAF-AH and PAF-R were designed based on the published sequences and are listed in .]\j!s9|, 百拇医药
fig.ommtted]\j!s9|, 百拇医药
Primer and probe sequences of PAF-AH and PAF-R]\j!s9|, 百拇医药
Statistical analysis.
Incidence of shock and mortality were compared using the 2 test. mRNA and antibody levels were compared using the Student’s t test. Differences were considered significant at P < 0.05.?9m)1-, 百拇医药
RESULTS?9m)1-, 百拇医药
Shock induced by antigen vaccination.?9m)1-, 百拇医药
No clinical shock reactions were observed after primary injection for any of the tested autoantigens (GAD65, ins-B, and hsp65) and diabetes-irrelevant antigens (OVA, KLH, TT, and PLP). However, after the booster injection with HEL, all tested NOD mice (11/11) had a clinical picture of shock and three mice (27%) subsequently died . This shock occurred within the first hour after injection and was characterized by pilo-erection; prostration; erythema of the tail, ears, and footpads; and dyspnea with shallow breathing. In the case of recovery, it occurred within another hour. The severity of these symptoms did not depend on the nature of the adjuvant because similar results were obtained when NOD mice where primed and rechallenged with HEL suspended in incomplete Freund’s adjuvant (shock in five of five mice). No signs of shock were observed in BALB/c (n = 9) or C57BL/6 (n = 9) mice. Booster injections with all other antigens were also uneventful in all tested mice.
fig.ommttedq[@}, 百拇医药
Shock incidence and severity in different HEL-immunized mouse strains. HEL, HEL-sensitized; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated. No signs of shock; reversible shock; lethal shock. **P < 0.01 concerning mortality. n = 9–17 mice per group.q[@}, 百拇医药
Treatment of NOD mice with Th2 enhancing 1{alpha} ,25(OH)2D3 exacerbated the previous observations of shock after the HEL booster. Whereas all (n = 17) 1,25(OH)2D3-treated NOD mice showed severe clinical signs of shock, 82% never recovered and died (P < 0.01 compared with vehicle-treated NOD mice). This enhancement of sensitivity to shock by 1{alpha} ,25(OH)2D3 was also observed in BALB/c mice. Here, in contrast to the symptom-free control BALB/c mice (n = 9), all 1,25(OH)2D3-treated BALB/c mice (n = 9) showed clinical signs of shock, although not life-threatening shock. This exacerbation by treatment with 1,25(OH)2D3 was not observed in C57BL/6 mice (n = 9), which all remained shock free .
Th2 shift parallels shock.-], 百拇医药
Because the clinical picture was compatible with a state of anaphylaxis, rescue treatment with corticosteroids was attempted. As shown in , pretreatment of NOD mice with 50 µg dexamethasone, injected intraperitoneally 48, 24, and 2 h before the HEL booster, rescued all NOD mice—control as well as 1{alpha} ,25(OH)2D3-treated (n = 8 for each group). This result suggests involvement of the immune system in the shock phenomenon. To further investigate the mechanism involved, antibody production was measured. Therefore, HEL-specific serum IgG1 and IgG2a and total serum IgE levels of the NOD and C57BL/6 mice, treated with 1{alpha} ,25(OH)2D3 or vehicle, were measured 3 weeks after the first HEL immunization (just before HEL rechallenge).-], 百拇医药
fig.ommtted-], 百拇医药
Effect of rescue treatment with dexamethasone on shock incidence and severity in NOD mice. HEL, HEL-sensitized; HEL+DEX, HEL-sensitized, rescued with dexamethasone; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated; HEL+D3+DEX, HEL-sensitized, 1,25(OH)2D3-treated, rescued with dexamethasone; No signs of shock; reversible shock; lethal shock. n = 8 mice per group.
No significant differences were found for Th1-induced HEL-specific IgG2a antibodies between control NOD and C57BL/6 mice, and no differences were found in the 1{alpha} ,25(OH)2D3-treated groups . Levels of HEL-specific IgG1 from NOD mice were very high in all HEL-immunized mice, irrespective of 1{alpha} ,25(OH)2D3 administration. Levels in NOD mice were higher compared with C57BL/6 mice, in vehicle- as well as in 1{alpha} ,25(OH)2D3-treated mice, although no significant difference was reached (). HEL-specific IgE antibodies were below the detection limit in our ELISA method (data not shown). However, total IgE levels, which were undetectable in the sera of control and CFA-injected NOD and C57BL/6 mice, were elevated in all HEL-immunized mice. Interestingly, total IgE levels were significantly higher in NOD mice than in C57BL/6 controls (P < 0.01; ). Moreover, this high IgE level in NOD mice was further increased by treatment with 1,25(OH)2D3 [P < 0.05 in 1,25(OH)2D3-treated NOD mice compared with control NOD mice; ], a phenomenon not observed in C57BL/6 mice.
fig.ommttedu/, http://www.100md.com
Impact of HEL immunization on antibody production. HEL-specific IgG2a (A), HEL-specific IgG1 (B), and total IgE (C) levels in sera from NOD mice and C57BL/6 mice , either not treated (HEL) or treated with 1,25(OH)2D3 (HEL+D3). *P < 0.05, **P < 0.01. n = 8 mice per group. OD, optical density.u/, http://www.100md.com
To further investigate the possible involvement of cytokines in this IgE response, cytokine mRNA levels specifically produced by Th1 or Th2 cells were quantified by real-time RT-PCR. In analogy to the serum antibody measurements, mRNA levels were quantified 3 weeks after the first HEL immunization in the spleen of NOD versus C57BL/6 mice, either 1{alpha} ,25(OH)2D3- or vehicle-treated. IFN- mRNA was significantly downregulated by treatment with 1{alpha} ,25(OH)2D3 in the HEL-immunized NOD mice (P < 0.05, ). Lower values of this typical Th1 cytokine were also seen in the 1,25(OH)2D3-treated C57BL/6 mice compared with their nontreated controls, although the difference did not reach significance. However, no difference was observed between the two strains with regard to IFN- mRNA . In contrast to the downregulated IFN-, IL-13 mRNA levels were dramatically upregulated by 1{alpha} ,25(OH)2D3 in the HEL-immunized NOD mice (P < 0.0001), an effect that was not observed in the C57BL/6 mice. Moreover, for this Th2-specific cytokine, an obvious strain difference was seen between HEL-immunized NOD and C57BL/6 mice (P < 0.05), a difference that was even more pronounced during treatment with 1{alpha} ,25(OH)2D3 (P < 0.0001, ). No differences in IL-4 or in the macrophage-produced cytokines IL-1, IL-6, IL-12, and TNF-{alpha} mRNA levels were observed (data not shown).
fig.ommtteds', 百拇医药
Impact of HEL immunization on cytokine expression. IFN- (A) and IL-13 (B) mRNA levels in spleen of NOD mice versus C57BL/6 mice. Control equals nonsensitized mice. HEL, HEL-sensitized mice; HEL+D3, HEL-sensitized and 1,25(OH)2D3-treated mice. *P < 0.05. Each bar is the mean of n = 8–15 mice (±SE).s', 百拇医药
Finally, NOD-scid mice lacking T- and B-cells were primed and rechallenged with HEL/CFA. Interestingly, in these mice (n = 6), no symptoms of shock were observed, conclusively demonstrating the key role of T-cells in the observed phenomenon.s', 百拇医药
Involvement of platelet activation in shock.s', 百拇医药
Because the clinical picture of lethal shock was accompanied by hypercoagulation, compatible with anaphylaxis leading to death by disseminated intravasal coagulation, we questioned the involvement of platelet activation in induced shock. We investigated if factors involved in this pathway showed an abnormal expression profile in the NOD mouse or were influenced by 1{alpha} ,25(OH)2D3. Therefore, mRNA levels for PAF-R and PAF-AH, the enzyme most responsible for degradation of PAF, were quantified by real-time RT-PCR in the spleens of NOD and C57BL/6 mice 3 weeks after the first footpad injection with HEL.
Interestingly, a clear inter-strain difference in the expression of PAF-R mRNA was observed, with 2.5-fold higher levels in NOD mice than in C57BL/6 mice (P < 0.05, ). Additionally, 1{alpha} ,25(OH)2D3 administration further increased the expression PAF-R mRNA in NOD as well as C57BL/6 mice by 1.5- and 2.5-fold, respectively (P < 0.05, ). These results, showing a regulation of the PAF-R in the analyzed system, are highly indicative for an important role of the PAF pathway in induction and sensitivity for anaphylaxis.2qw, 百拇医药
fig.ommtted2qw, 百拇医药
Impact of HEL immunization on PAF-R (A) and PAF-AH (B) mRNA levels in spleens of NOD or C57BL/6 mice not treated (HEL) or treated with 1,25(OH)2D3 (HEL+D3). *P < 0.05; **P < 0.01. Each bar is the mean of n = 8–15 mice (±SE).2qw, 百拇医药
The mRNA expression of PAF-AH in the spleens of HEL-immunized NOD mice was 1.5-fold higher than that in C57BL/6 mice (P < 0.01). 1{alpha} ,25(OH)2D3 treatment did not influence the PAF-AH mRNA levels. A minor interstrain difference remained after 125(OH)2D3 treatment; however, this difference did not reach significance
To conclusively demonstrate the involvement of the platelet activation pathway in the shock phenomenon, we attempted to rescue the HEL-immunized NOD mice from lethal shock by administration of a PAF-R antagonist. Indeed, mortality was completely abrogated by administration of a single dose of 500 µg of the PAF-R antagonist WEB2086 10 min before the HEL booster in all HEL-immunized NOD mice, irrespective of 1{alpha} ,25(OH)2D3 treatment . Only mild nonlethal shock symptoms were observed in 75% of the rescued mice; in each group, shock was completely prevented in two of eight mice, whereas the other six developed milder shock symptoms, from which they all recovered .g, http://www.100md.com
fig.ommttedg, http://www.100md.com
Effect of rescue treatment with WEB2086 on shock incidence and severity in NOD mice. HEL, HEL-sensitized; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated; HEL+D3+WEB, HEL-sensitized, 1,25(OH)2D3-treated, rescued with WEB2086; HEL+WEB, HEL-sensitized, rescued with WEB2086. No signs of shock; reversible shock; , lethal shock. *P < 0.05 concerning disease incidence. n = 8 mice per group.
DISCUSSION+'3, 百拇医药
Major advances have recently been made in better understanding the pathogenesis of various autoimmune diseases. Data obtained from animal models of human type 1 diabetes and MS suggest that, in these diseases, a pivotal role in immune self-destruction is played by Th1 cells (17). Efforts have thus been made to find ways of promoting T-helper differentiation toward a Th2 rather than Th1 subtype, thereby arresting the autoimmune destructive process (5). It has been observed that a protective switch of T-helper differentiation could be obtained by vaccination with disease-triggering autoantigens (18,19). Moreover, this strategy has been extrapolated to human MS (10) and is about to be adopted in human type 1 diabetes (8,20). However, initial enthusiasm was tempered by the observation that vaccination with self-antigens could elicit anaphylactic shock in the context of a disturbed immune system (11,12). This anaphylactic reaction could be triggered by exactly the same T-cell differentiation shift that provided protection against autoimmunity (21). In contrast to a Th1-mediated autoimmune attack, allergic disease is mainly Th2 dependent (22) and is largely mediated by IgE (22,23). Aggressive allergic reactions to encephalitogenic antigens were also observed when the vaccination approach was tested in human MS (12,24). The benefit of protection against an autoimmune disease could therefore be hampered by the allergic side effects.
NOD mice are animals that have a special immune system, with particularities of both T-cells and antigen-presenting cells (2). Although their most known immune feature is the Th1-driven destruction of pancreatic islets (4), literature data about their Th2 activity are conflicting (25,26). The initial aim of our study was to check the safety of antigenic vaccination in this particular strain with respect to anaphylaxis. The set of antigens tested included diabetes-relevant autoantigens (GAD65, mouse insulin, and hsp65) and diabetes-irrelevant self (PLP) or nonself (HEL, OVA, KLH, and TT) antigens. The behavior of NOD mice to vaccination was compared with that of two other mouse strains: C57BL/6 and BALB/c mice. To force T-cell differentiation toward a Th2 setting, we submitted groups of mice from the three strains to therapy with 1{alpha} ,25(OH)2D3. The active form of vitamin D is known as a powerful modulator of the immune system (27). The 1{alpha} ,25(OH)2D3-induced shift toward Th2 differentiation can even prevent the onset of Th1-mediated autoimmune diseases, such as diabetes in NOD mice (28) or EAE in SJL/J mice (29).
The only antigen that provoked anaphylactic reactions in our experiments was HEL. However, mice from various strains behaved in different ways. C57BL/6 mice were completely resistant to HEL-induced anaphylaxis, irrespective of 1{alpha} ,25(OH)2D3 administration. This outcome was not unexpected because C57BL/6 mice are known for having a predominantly Th1-driven immune response, though not accompanied by autoimmune phenomena (30). Although T-cell lymphocyte differentiation is mostly Th2-directed in BALB/c mice (30), HEL vaccination by itself did not evolve to shock. When the immune system was further stressed toward Th2 responses by 1{alpha} ,25(OH)2D3 administration, all BALB/c mice nevertheless developed a mild reversible form of shock. Unexpectedly, NOD mice, known for their Th1-mediated immune reactivity, also developed shock consecutive to HEL sensitization. Moreover, shock appeared in NOD mice even without any modulation of the immune system with 1{alpha} ,25(OH)2D3 and was rather aggressive (even lethal for certain animals). 1{alpha} ,25(OH)2D3 administration further increased mortality in HEL-sensitized NOD mice.
We were surprised by the strain-selective anaphylactic shock induced by HEL in NOD mice. This mouse strain is indeed known to react through Th1-mediated mechanisms (2–4,25) and was moreover described as having a defect in antigen presentation (31,32). An exacerbation of anaphylaxis caused by the administration of 1{alpha} ,25(OH)2D3 was also not immediately clear because previous studies suggested that a 1{alpha} ,25(OH)2D3-dependent differentiation shift toward Th2 takes place exclusively in the presence of diabetes-relevant autoantigens (33). We decided therefore to search for the mechanisms underlying the strain-specific behavior of the NOD immune system.4k/, 百拇医药
Complete rescue by therapy with dexamethasone demonstrated undoubtedly that HEL-induced shock in NOD mice depended on immune effectors. Furthermore, the complete absence of symptoms in NOD-scid mice lacking T- and B-cells (34) proved the indispensable role of T-cells in HEL-induced shock. Cytokine modifications caused by 1{alpha} ,25(OH)2D3 in NOD mice were partially suggestive for a Th1 to Th2 shift, with a decrease in Th1-secreted IFN-{gamma} and a concomitant increase in Th2-specific IL-13. However, no differences in IL-12 and IL-4 levels were found between the 1{alpha} ,25(OH)2D3-treated and nontreated HEL-sensitized NOD mice. Thus, these changes did not completely overlap the 1{alpha} ,25(OH)2D3-dependent modifications found when the immune system was activated by autoantigens (33), suggesting antigen-specific immune modulation by 1{alpha} ,25(OH)2D3. Comparison of cytokine mRNA spectra between HEL-immunized shock-sensitive NOD mice and shock-resistant C57BL/6 mice showed differences in IL-13 mRNA levels, which were further accentuated by 1{alpha} ,25(OH)2D3 administration. Therefore, our data supported that the cytokine has a critical role in the particular behavior of NOD mice toward HEL sensitization. IL-13 is a Th2-specific cytokine closely related to IL-4 (35) and synthesized in both humans and mice (36). IL-13 has been alleged to be importantly involved in Th2-initiated allergic reactions, including anaphylaxis (37). The significant difference in serum IgE levels between the two mouse strains provided evidence of IgE-mediated anaphylactic shock in NOD mice. IgE is an immunoglobulin involved in most allergic reactions (13,38). The parallelism between IL-13 mRNA levels and serum IgE levels assessed in our mice stresses a causal relationship between the two immune parameters. Direct stimulation of IgE synthesis by IL-13 has been actually described (38). Certain NOD mice also displayed higher levels of Th2-dependent IgG1. IgG1 may by itself induce anaphylactic shock in mice (11,13).
PAF is an important mediator of immune, hemostatic, and allergic reactions (39). This bioactive phospholipid can be regarded as a cytokine because of its multiple actions on a wide range of cells (including neutrophils, monocytes, and platelets) and tissues that possess specific membrane receptors (PAF-R) (40). The acute inflammatory effects of PAF are largely due to the stimulation of neutrophilic granulocytes as the first line of defense (40). Exaggerated synthesis of PAF or defective mechanisms of PAF inactivation can however lead to pathologic inflammation, shock, and thrombosis (39). There are data in the literature suggesting significant interference of 1,25(OH)2D3 with PAF actions (41) or metabolism (42). Because 1,25(OH)2D3 had such a powerful impact on shock incidence and severity, we suspected an interference of PAF in HEL-induced shock. PAF-R mRNA levels were significantly higher in NOD mice, pleading in favor of an increased sensitivity to PAF inflammatory actions in this strain. In concordance with other studies (43), 1,25(OH)2D3 therapy caused a further increase in the PAF-R expression, a phenomenon that might partly explain the increase in shock severity. Surprisingly, PAF-AH mRNA levels in HEL-sensitized NOD mice were significantly higher than those in C57BL/6 mice. PAF-AH is the main enzyme involved in PAF degradation. The increase in PAF-AH mRNA levels after HEL sensitization could be the expression of a natural feedback reaction against exaggerated PAF synthesis (44). Contrary to another study (42), 1,25(OH)2D3 therapy did not significantly inhibit PAF-AH mRNA expression in HEL-sensitized NOD mice. However, the minor interstrain difference of PAF-AH levels was no longer significant in the 1,25(OH)2D3-treated groups. The direct proof of PAF involvement in the etiopathogenesis of strain-specific HEL-induced shock was obtained when rescue therapy with a PAF-R antagonist (WEB2086) showed to be efficient in preventing shock or diminishing its severity. The activation of the Th2 arm of the immune attack is probably linked to the activation of the PAF system. IgE, for example, can undeniably cause direct stimulation of PAF synthesis by various cells, such as mastocytes (41). Moreover, a recent article (45) suggested that murine antigen-induced anaphylaxis is primarily mediated by the PAF pathway and not by the classic IgE pathway.
Finally the intriguing question remains why HEL induces anaphylaxis in the NOD mouse model, as opposed to the other antigens tested. An extensive GenBank search did not reveal any peptide homology between HEL and any of the known diabetes-relevant autoantigens, not excluding, however, a possible similarity with an as yet undefined autoantigen.5iawd, 百拇医药
In conclusion, our data clearly demonstrate the appearance of a severe Th2-biased immune reaction after challenge with a widely used control antigen in a mouse strain known mainly for its genetic propensity toward Th1-mediated autoimmune destruction of ß-cells. Strain specificity of this reaction points out that immune systems with a disturbed genetic background may reply unpredictably to an immune challenge, even with "innocent" antigens. Autoimmunity should therefore not be simply viewed as an isolated particularity of the immune response but rather as a general disturbance of the immune system. It is currently accepted that Th1-oriented autoimmunity is involved in the pathogenesis of human type 1 diabetes (1–5). Shifting the activation of the immune system toward the Th2 differentiation pathway has been proven efficient in preventing diabetes in animal models (2,3,5) and might be regarded as an interesting alternative to type 1 diabetes prevention in humans (5,9). The immune system of type 1 diabetic patients may, however, manifest changes beyond the local islet-destructive effect (46). Our data complete the conflicting experience already existing with vaccination strategies in EAE and human MS (11,21,24). Extreme caution is therefore needed when adopting immune-modulating strategies, taking into consideration that a shift in T-helper differentiation might always represent a transfer of risk from a Th1-mediated type IV delayed immune attack to a Th2-mediated anaphylactic reaction, especially in situations with an already-activated Th2 system, such as a coincidental infection or allergy.
ACKNOWLEDGMENTS$z|8, 百拇医药
This work was supported by the Flemish Research Foundation (Fonds voor Wetenschappelijk Onderzoek [FWO] grant G.0084.02), the Interuniversity Attraction Poles (IUAP/P5/03/20), the European Foundation for the Study of Diabetes, the Flemish Governments of Research and Education (Geconerteerde Onderzoeksactic [GOA] 99/10), and an FWO fellowship for Chantal Mathieu.$z|8, 百拇医药
The expert technical assistance of Jos Laureys is gratefully acknowledged.$z|8, 百拇医药
REFERENCES$z|8, 百拇医药
Yoon JW, Jun HS: Cellular and molecular pathogenic mechanisms of insulin-dependent diabetes mellitus. Ann N Y Acad Sci928 :200 –211,2001$z|8, 百拇医药
Adorini L, Gregori S, Harrison LC: Understanding autoimmune diabetes: insights from mouse models. Trends Mol Med8 :31 –38,2002$z|8, 百拇医药
Rabinovitch A: An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes Metab Rev14 :129 –151,1998$z|8, 百拇医药
Wong FS, Janeway CA Jr: Insulin-dependent diabetes mellitus and its animal models. Curr Opin Immunol11 :643 –647,1999
Casares S, Brumeanu TD: Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies. Curr Mol Med1 :357 –378,2001-e7&(, 百拇医药
Kundig TM, Bachmann MF, Ohashi PS, Pircher H, Hengartner H, Zinkernagel RM: On T cell memory: arguments for antigen dependence. Immunol Rev150 :63 –90,1996-e7&(, 百拇医药
Foucras G, Gallard A, Coureau C, Kanellopoulos JM, Guery JC: Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with Th2 phenotype acquisition in vivo. J Immunol168 :179 –187,2002-e7&(, 百拇医药
Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen IR: ß-Cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind phase II trial. Lancet358 :1749 –1753,2002-e7&(, 百拇医药
Ramiya VK, Lan MS, Wasserfall CH, Notkins AL, Maclaren NK: Immunization therapies in the prevention of diabetes. J Autoimmun10 :287 –292,1997-e7&(, 百拇医药
Willenborg DO, Staykova MA: Approaches to the treatment of central nervous system autoimmune disease using specific neuroantigen. Immunol Cell Biol76 :91 –103,1998
Pedotti R, Mitchell D, Wedemeyer J, Karpuj M, Chabas D, Hattab EM, Tsai M, Galli SJ, Steinman L: An unexpected version of horror autotoxicus: anaphylactic shock to a self-peptide. Nat Immunol2 :216 –222,20010|pjki, 百拇医药
Kappos L, Comi G, Panitch H, Oger J, Antel J, Conlon P, Steinman L, The Altered Peptide Ligand in Relapsing MS Study Group: Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. Nat Med6 :1176 –1182,20000|pjki, 百拇医药
Harada M, Nagata M, Takeuchi M, Makino S, Aburahi S: High susceptibility of cataract Shionogi (CTS) mice to passive anaphylactic shock mediated by allogeneic IgE and IgG1 monoclonal antibodies. Immunol Invest20 :305 –315,19910|pjki, 百拇医药
Hurtenbach U, Lier E, Adorini L, Nagy ZA: Prevention of autoimmune diabetes in non-obese diabetic mice by treatment with a class II major histocompatibility compex-blocking peptide. J Exp Med177 :1499 –1504,19930|pjki, 百拇医药
Pozilli P, Signore A, Williams AJ, Beales P: NOD mouse colonies around the world: recent facts and figures. Immunol Today14 :193 –196,1993
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods25 :386 –401,2001n&, http://www.100md.com
Singh VK, Mehrotra S, Agarwal SS: The paradigm of Th1 and Th2 cytokines: its relevance to autoimmunity and allergy. Immunol Res20 :147 –161,1999n&, http://www.100md.com
Parish NM, Hutchings PR, O’Reilly L, Quartey-Papafio R, Healey D, Ozegbe P, Cooke A: Tolerance induction as a therapeutic strategy for the control of autoimmune endocrine disease in mouse models. Immunol Rev144 :269 –300,1995n&, http://www.100md.com
Bach JF, Chatenoud L: Tolerance to islet autoantigens in type 1 diabetes. Annu Rev Immunol19 :131 –161,2001n&, http://www.100md.com
Simone EA, Wegmann DR, Eisenbarth GS: Immunologic "vaccination" for the prevention of autoimmune diabetes (type 1A). Diabetes Care22 (Suppl. 2) :B7 –B15,1999n&, http://www.100md.com
Silverstein AM: Autoimmunity versus horror autotoxicus: the struggle for recognition. Nat Immunol2 :279 –281,2001n&, http://www.100md.com
Broide DH: Molecular and cellular mechanisms of allergic disease. J Allergy Clin Immunol108 :S65 –S71,2001
Kay AB: Allergy and allergic diseases: first of two parts. N Engl J Med344 :30 –37,2001jmd^, http://www.100md.com
Wiendl H, Neuhaus O, Kappos L, Hohlfeld R: Multiple sclerosis: current view of failed and discontinued clinical trials of drug treatment. Nervenarzt71 :597 –610,2000jmd^, http://www.100md.com
Hartemann AH, Richard MF, Boitard C: Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice. Clin Exp Immunol116 :225 –230,1999jmd^, http://www.100md.com
Schloot NC, Hanifi-Moghaddam P, Goebel C, Shatavi SV, Flohe S, Kolb H, Rothe H: Serum IFN-gamma and IL-10: levels are associated with disease progression in non-obese diabetic mice. Diabetes Metab Res Rev18 :64 –70,2002jmd^, http://www.100md.com
Mathieu C, Adorini L: The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents. Trends Mol Me8 :174 –179,2002jmd^, http://www.100md.com
Mathieu C, Laureys J, Sobis H, Vandeputte M, Waer M, Bouillon R: 1,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice. Diabetes41 :1491 –1495,1992jmd^, http://www.100md.com
Branisteanu DD, Waer M, Sobis H, Marcelis S, Vandeputte M, Bouillon R: Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 alpha, 25-(OH)2D3. J Neuroimmunol6 :151 –160,1995
Hayashi T, Hasegawa K, Nakai S, Hamachi T, Adachi Y, Yamauchi Y, Maeda K: Bronchial lesions of the late asthmatic response in Balb/c and C57BL/6 mice. J Comp Pathol125 :208 –213,2001re@qgv, 百拇医药
Strid J, Lopes L, Marcinkiewicz J, Petrovska L, Nowak B, Chain BM, Lund T: A defect in bone marrow derived dendritic cell maturation in the nonobese diabetic mouse. Clin Exp Immunol123 :375 –381,2001re@qgv, 百拇医药
Piganelli JD, Martin T, Haskins K: Splenic macrophages from the NOD mouse are defective in the ability to present antigen. Diabetes47 :1212 –1218,1998re@qgv, 百拇医药
Overbergh L, Decallonne B, Waer M, Rutgeerts O, Valckx D, Casteels KM, Laureys J, Bouillon R, Mathieu C: 1alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524–543). Diabetes49 :1301 –1307,2000re@qgv, 百拇医药
Shultz L, Schweitzer P, Christianson S, Gott B, Schweitzer I, Tennent B, McKenna S, Mobraaten L, Rajan T, Greiner D, Leiter E: Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol154 :180 –191,1995
Zurawski G, de Vries JE: Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today15 :19 –26,199488%, 百拇医药
Brombacher F: The role of interleukin-13 in infectious diseases and allergy. Bioessays22 :646 –656,200088%, 百拇医药
Fallon PG, Emson CL, Smith P, McKenzie AN: IL-13 overexpression predisposes to anaphylaxis following antigen sensitization. J Immunol166 :2712 –2716,200188%, 百拇医药
Corry DB, Kheradmand F: Induction and regulation of the IgE response (Review Article) Nature402 (Suppl. 6760) :B18 –B23,199988%, 百拇医药
Ayala A, Chaudry IH: Platelet activating factor and its role in trauma, shock, and sepsis. New Horiz4 :265 –275,199688%, 百拇医药
Snyder F: Platelet-activating factor and its analogs: metabolic pathways and related intracellular processes. Biochim Biophys Acta1254 :231 –249,199588%, 百拇医药
Chen SF, Ruan YJ: 1 alpha,25-Dihydroxyvitamin D3 decreases scalding- and platelet-activating factor-induced high vascular permeability and tissue oedema. Pharmacol Toxicol76 :365 –367,199588%, 百拇医药
Narahara H, Miyakawa I, Johnston JM: The inhibitory effect of 1,25-dihydroxyvitamin D3 on the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages. J Clin Endocrinol Metab80 :3121 –3126,199588%, 百拇医药
Muller E, Dupuis G, Turcotte S, Rola-Pleszczynski M: Human PAF receptor gene expression: induction during HL-60 cell differentiation. Biochem Biophys Res Commun181 :1580 –1586,199188%, 百拇医药
Stafforini DM, McIntyre TM, Zimmerman GA, Prescott SM: Platelet-activating factor acetylhydrolases. J Biol Chem272 :17895 –17898,199788%, 百拇医药
Strait RT, Morris SC, Yang M, Qu XW, Finkelman FD: Pathways of anaphylaxis in the mouse. J Allergy Clin Immunol109 :658 –668,200288%, 百拇医药
Friday RP, Trucco M, Pietropaolo M: Genetics of type 1 diabetes mellitus. Diabetes Nutr Metab12 :3 –26,1999(Lut Overbergh Brigitte DecallonneDumitru D. Branisteanu Dirk Valck Ahmad Kasran Roger Bouillon and C)
2 Clinic of Endocrinology, University Hospital "St. Spiridon," University of Medicine and Pharmacy "Gr. T. Popa," Iasi, Romaniah%tj, 百拇医药
3 Laboratory for Immunology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgiumh%tj, 百拇医药
ABSTRACTh%tj, 百拇医药
Type 1 diabetes in NOD mice can be prevented through autoantigen vaccination by shifting lymphocyte differentiation toward a T-helper 2 (Th2) response. However, in other models of autoimmunity, this approach may be accompanied by unexpected triggering of Th2-dependent anaphylactic shock. To test the safety of vaccination therapy in the NOD mouse model, we evaluated the effects of immunization with a wide battery of antigens in NOD, BALB/c, and C57BL/6 mice. Surprisingly, a nondiabetogenic antigen, hen egg white lysozyme, induced severe shock exclusively in NOD mice (shock in 11 of 11 mice, lethal in 3 mice). Shock severity was further increased by a more pronounced Th2 setting generated by 1{alpha} ,25(OH)2D3 administration (17 of 17 mice, lethal in 14 mice, P < 0.0001). Pretreatment with dexamethasone resulted in full rescue, indicating an immune-mediated mechanism. Serum IgE levels and Th1/Th2 cytokine profile analysis showed that the shock phenomenon was paralleled by a Th2 response. mRNA expression of platelet-activating factor receptor (PAF-R) was significantly higher in NOD mice (P < 0.01) and was further increased by 1{alpha} ,25(OH)2D3. Pretreatment with WEB2086 (PAF-R antagonist) again protected all mice from lethal shock, indicating PAF as an anaphylaxis effector. In conclusion, in NOD mice, vaccination leading to a Th2 immune shift can result in a lethal anaphylactic reaction.
Type 1 diabetes is caused by immune destruction of insulin-producing ß-cells in the pancreatic islets of Langerhans (1). In the NOD mouse, a widely used animal model for the study of type 1 diabetes, lymphocytic infiltration into the pancreatic islets (insulitis) is the primary lesion that eventually progresses to overt diabetes. Evidence is accumulating that type 1 diabetes is mediated by T-helper 1 (Th1) cells and that protection or prevention of the disease can be achieved by shifting the immune response toward Th2 activation (2,3).qc'r, 百拇医药
Several diabetogenic autoantigens have been described, such as GAD, insulin, and heat shock protein, in both animal models and human disease (4,5). Furthermore, epitope spreading and molecular mimicry is enlarging their spectrum during disease evolution (5). The biochemical properties of antigens together with the genetic background of the immune system significantly influence the type (Th1 or Th2) of immune activation (6). Many studies have investigated the effects of administration of diabetes relevant autoantigens or their immunodominant peptides in the NOD mouse model. Most studies convincingly demonstrate a prevention or delayed onset of disease, associated with a shift toward a more Th2-biased response (5,7). Recently, also in type 1 diabetic patients, peptide treatment with specific autoantigens has been shown to prevent or delay the disease and was concluded to be safe (8).
This vaccination-triggered shift from autoreactive Th1 toward Th2 cells has not only been observed in NOD mice (9) but also in experimental autoimmune encephalomyelitis (EAE), another Th1-mediated autoimmune disease (10). Nevertheless, the administration of self-antigen in the EAE model has lead to unforeseen consequences of Th2 activation. Indeed, vaccination with the myelin proteolipid peptide PLPp(139–151) in SJL/J mice under certain conditions, essentially depending on the time point of peptide rechallenge, unexpectedly resulted in lethal anaphylaxis (11). Also in the human situation, a recent phase II trial in which multiple sclerosis (MS) patients received an altered peptide ligand from a myelin basic protein epitope, repeated injections with the peptide induced immediate hypersensitivity reactions in 9% of the subjects (12). These reports suggest that an autovaccine-triggered immune response may produce extremely undesirable allergic reactions. To what extent anaphylaxis depends on the genetic background, the antigen type, or the timing of vaccination is not yet completely known.
The aim of the present study was to test whether vaccination in NOD mice, animals having an immune system genetically prone to destroy pancreatic islets through Th1-mediated mechanisms, is perfectly safe or whether life-threatening reactions can be induced by manipulating the immune response. Indeed, the NOD mouse strain also displays immune system particularities beyond Th1-dependent autoimmune islet destruction because they seem to be highly susceptible to anaphylactic shock under certain specified conditions (13,14). NOD mice might therefore behave differently from other mouse strains in certain situations of immune challenge (13). To investigate whether vaccination could induce lethal shock in NOD mice, we tested a wide panel of antigens. We compared the effects of several diabetes-relevant autoantigens and diabetes-irrelevant peptides or proteins in NOD mice to the effects obtained in two other mouse strains (BALB/c and C57BL/6 mice), in which a Th1-activated immune system is absent. Based on our previous findings, we also treated mice from various strains with an immunomodulatory agent known to favor an autoantigen-specific Th2 shift in NOD mice, namely 1,25(OH)2D3, and examined its effect on the safety profile of vaccination.
RESEARCH DESIGN AND METHODS1}afv{}, http://www.100md.com
Animals.1}afv{}, http://www.100md.com
NOD mice were originally obtained from Prof. C.Y. Wu in 1989 and further bred in our animal house (Proefdierencentrum, Leuven, Belgium), kept under semi-barrier conditions (15). At the time of the study, diabetes incidence by the age of 200 days in our stock colony was 72% in female mice and 25% in male mice. Nondiabetic mice aged 7–9 weeks of both sexes were used in the experiments. BALB/c and C57BL/6 mice were purchased from Harlan (Horst, the Netherlands). NOD/LtSz-scid/scid (NOD-scid) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and kept under specific pathogen-free conditions. Mice were fed regular diet and water ad libitum. Animals receiving 1{alpha} ,25(OH)2D3 or vehicle were fed a low-calcium vitamin D–replete diet (0.2% calcium, 1% phosphate, 2,000 units vitamin D/kg; Hope Farms, Woerden, the Netherlands).1}afv{}, http://www.100md.com
Proteins and peptides.
Hen egg white lysozyme (HEL) (94% purity), ovalbumin (OVA) (Grade V, 98% purity), keyhole limpet hemocyanine (KLH), bovine insulin B (ins-B) chain, and PLP peptide (amino acids 135–151) were purchased from Sigma (Bornem, Belgium). GAD65 (p524–542) (SRLSKVAPVIKARMMEYGTT), heat shock protein (hsp)-65 peptide (PALDSLTPANED), and tetanus toxin (TT) peptide (p830–843) (QYIKANSKIGITE) contained a COOH-terminal amidation (CONH2) group and were >75% pure (Eurogentec, Luik, Belgium).|5, 百拇医药
Vaccination and intervention regimens.|5, 百拇医药
The 8-week-old NOD, BALB/c, and C57BL/6 mice were immunized by injection of 100 µg antigen (HEL, GAD65, ins-B, hsp65, PLP, OVA, KLH, and TT) emulsified at a 1:1 concentration in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (Difco Laboratories, Detroit, MI) in the hind footpads. NOD-scid mice were injected with 100 µg HEL antigen suspended in CFA. All mice were reinjected with the same antigen 3 weeks later in a similar manner.
NOD, BALB/c, and C57BL/6 mice were treated with 1{alpha} ,25(OH)2D3 from 3 weeks of age until the end of the experiment. The drug was suspended in arachis oil and administered at a dose of 5 µg/kg i.p. every 2 days. Control NOD, BALB/c, and C57BL/6 mice received the treatment vehicle.6., 百拇医药
Dexamethasone (50 µg dissolved in 0.9% NaCl; Aacidexam, NV Organon, Oss, the Netherlands) was injected intraperitoneally in NOD mice 48, 24, and 2 h before the booster injection. WEB2086 (platelet-activating factor receptor [PAF-R] antagonist; Boehringer Mannheim, Mannheim, Germany; a gift from Prof. Vermylen, University of Leuven, Leuven, Belgium), 500 µg dissolved in PBS by sonication, was injected intraperitoneally in NOD mice 10 min before the HEL booster injection.6., 百拇医药
Follow-up of mice.6., 百拇医药
Clinical evolution and survival rate after sensitization with various peptides were monitored in different mouse strains. Shock was characterized by pilo-erection; prostration; erythema of the tail, ears, and footpads; and dyspnea with shallow breathing. Serum for antibody measurement and spleens for quantification of mRNA levels were collected before immunization and again before booster administration.
Measurement of antibody production.{1(!(kz, 百拇医药
HEL-specific IgG1, IgG2a, and IgE antibodies were measured in mouse sera by enzyme-linked immunosorbent assay (ELISA). Briefly, for IgG1 and IgG2a ELISA, 96-well microtiter plates were coated overnight with HEL (10 µg/ml). Samples for IgG1 and IgG2a measurement were diluted at 1:3,000 and 1:800, respectively. Bound IgG1 and IgG2a were detected using biotinylated anti-mouse IgG1 or anti-mouse IgG2a, respectively. The plates were further processed with horseradish peroxidase–conjugated streptavidin and tetramethylbenzidine as substrate. To measure HEL-specific IgE antibody, plates were coated with anti-mouse IgE antibody. After incubation with diluted mouse sera (1:20), bound IgE was incubated with HEL. The plates were then processed with peroxidase-conjugated anti-HEL antibody, and tetramethylbenzidine was used as a substrate. Plates were read at 450 nm on a MultiSkan ELISA reader (ThermoLab Systems, Brussels, Belgium).
Total IgE was measured by sandwich ELISA (BD Biosciences, Erembodegem, Belgium) according to the manufacturer’s instructions. Serum samples were analyzed in duplicate at a 1:10 dilution.]\j!s9|, 百拇医药
Real-time RT-PCR for cytokines, PAF-acetylhydrolase, and PAF-R.]\j!s9|, 百拇医药
Total RNA from spleen was extracted using the SV total RNA isolation system (Promega, Madison, WI). A 1-µg target RNA was reverse-transcribed as described previously (16).]\j!s9|, 百拇医药
Real-time quantitative RT-PCR was performed for ß-actin, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-, IL-12, -interferon (IFN-), IL-4, IL-13, PAF-acetylhydrolase (AH), and PAF-R as previously described (16). The primers and fluorogenic probe for PAF-AH and PAF-R were designed based on the published sequences and are listed in .]\j!s9|, 百拇医药
fig.ommtted]\j!s9|, 百拇医药
Primer and probe sequences of PAF-AH and PAF-R]\j!s9|, 百拇医药
Statistical analysis.
Incidence of shock and mortality were compared using the 2 test. mRNA and antibody levels were compared using the Student’s t test. Differences were considered significant at P < 0.05.?9m)1-, 百拇医药
RESULTS?9m)1-, 百拇医药
Shock induced by antigen vaccination.?9m)1-, 百拇医药
No clinical shock reactions were observed after primary injection for any of the tested autoantigens (GAD65, ins-B, and hsp65) and diabetes-irrelevant antigens (OVA, KLH, TT, and PLP). However, after the booster injection with HEL, all tested NOD mice (11/11) had a clinical picture of shock and three mice (27%) subsequently died . This shock occurred within the first hour after injection and was characterized by pilo-erection; prostration; erythema of the tail, ears, and footpads; and dyspnea with shallow breathing. In the case of recovery, it occurred within another hour. The severity of these symptoms did not depend on the nature of the adjuvant because similar results were obtained when NOD mice where primed and rechallenged with HEL suspended in incomplete Freund’s adjuvant (shock in five of five mice). No signs of shock were observed in BALB/c (n = 9) or C57BL/6 (n = 9) mice. Booster injections with all other antigens were also uneventful in all tested mice.
fig.ommttedq[@}, 百拇医药
Shock incidence and severity in different HEL-immunized mouse strains. HEL, HEL-sensitized; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated. No signs of shock; reversible shock; lethal shock. **P < 0.01 concerning mortality. n = 9–17 mice per group.q[@}, 百拇医药
Treatment of NOD mice with Th2 enhancing 1{alpha} ,25(OH)2D3 exacerbated the previous observations of shock after the HEL booster. Whereas all (n = 17) 1,25(OH)2D3-treated NOD mice showed severe clinical signs of shock, 82% never recovered and died (P < 0.01 compared with vehicle-treated NOD mice). This enhancement of sensitivity to shock by 1{alpha} ,25(OH)2D3 was also observed in BALB/c mice. Here, in contrast to the symptom-free control BALB/c mice (n = 9), all 1,25(OH)2D3-treated BALB/c mice (n = 9) showed clinical signs of shock, although not life-threatening shock. This exacerbation by treatment with 1,25(OH)2D3 was not observed in C57BL/6 mice (n = 9), which all remained shock free .
Th2 shift parallels shock.-], 百拇医药
Because the clinical picture was compatible with a state of anaphylaxis, rescue treatment with corticosteroids was attempted. As shown in , pretreatment of NOD mice with 50 µg dexamethasone, injected intraperitoneally 48, 24, and 2 h before the HEL booster, rescued all NOD mice—control as well as 1{alpha} ,25(OH)2D3-treated (n = 8 for each group). This result suggests involvement of the immune system in the shock phenomenon. To further investigate the mechanism involved, antibody production was measured. Therefore, HEL-specific serum IgG1 and IgG2a and total serum IgE levels of the NOD and C57BL/6 mice, treated with 1{alpha} ,25(OH)2D3 or vehicle, were measured 3 weeks after the first HEL immunization (just before HEL rechallenge).-], 百拇医药
fig.ommtted-], 百拇医药
Effect of rescue treatment with dexamethasone on shock incidence and severity in NOD mice. HEL, HEL-sensitized; HEL+DEX, HEL-sensitized, rescued with dexamethasone; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated; HEL+D3+DEX, HEL-sensitized, 1,25(OH)2D3-treated, rescued with dexamethasone; No signs of shock; reversible shock; lethal shock. n = 8 mice per group.
No significant differences were found for Th1-induced HEL-specific IgG2a antibodies between control NOD and C57BL/6 mice, and no differences were found in the 1{alpha} ,25(OH)2D3-treated groups . Levels of HEL-specific IgG1 from NOD mice were very high in all HEL-immunized mice, irrespective of 1{alpha} ,25(OH)2D3 administration. Levels in NOD mice were higher compared with C57BL/6 mice, in vehicle- as well as in 1{alpha} ,25(OH)2D3-treated mice, although no significant difference was reached (). HEL-specific IgE antibodies were below the detection limit in our ELISA method (data not shown). However, total IgE levels, which were undetectable in the sera of control and CFA-injected NOD and C57BL/6 mice, were elevated in all HEL-immunized mice. Interestingly, total IgE levels were significantly higher in NOD mice than in C57BL/6 controls (P < 0.01; ). Moreover, this high IgE level in NOD mice was further increased by treatment with 1,25(OH)2D3 [P < 0.05 in 1,25(OH)2D3-treated NOD mice compared with control NOD mice; ], a phenomenon not observed in C57BL/6 mice.
fig.ommttedu/, http://www.100md.com
Impact of HEL immunization on antibody production. HEL-specific IgG2a (A), HEL-specific IgG1 (B), and total IgE (C) levels in sera from NOD mice and C57BL/6 mice , either not treated (HEL) or treated with 1,25(OH)2D3 (HEL+D3). *P < 0.05, **P < 0.01. n = 8 mice per group. OD, optical density.u/, http://www.100md.com
To further investigate the possible involvement of cytokines in this IgE response, cytokine mRNA levels specifically produced by Th1 or Th2 cells were quantified by real-time RT-PCR. In analogy to the serum antibody measurements, mRNA levels were quantified 3 weeks after the first HEL immunization in the spleen of NOD versus C57BL/6 mice, either 1{alpha} ,25(OH)2D3- or vehicle-treated. IFN- mRNA was significantly downregulated by treatment with 1{alpha} ,25(OH)2D3 in the HEL-immunized NOD mice (P < 0.05, ). Lower values of this typical Th1 cytokine were also seen in the 1,25(OH)2D3-treated C57BL/6 mice compared with their nontreated controls, although the difference did not reach significance. However, no difference was observed between the two strains with regard to IFN- mRNA . In contrast to the downregulated IFN-, IL-13 mRNA levels were dramatically upregulated by 1{alpha} ,25(OH)2D3 in the HEL-immunized NOD mice (P < 0.0001), an effect that was not observed in the C57BL/6 mice. Moreover, for this Th2-specific cytokine, an obvious strain difference was seen between HEL-immunized NOD and C57BL/6 mice (P < 0.05), a difference that was even more pronounced during treatment with 1{alpha} ,25(OH)2D3 (P < 0.0001, ). No differences in IL-4 or in the macrophage-produced cytokines IL-1, IL-6, IL-12, and TNF-{alpha} mRNA levels were observed (data not shown).
fig.ommtteds', 百拇医药
Impact of HEL immunization on cytokine expression. IFN- (A) and IL-13 (B) mRNA levels in spleen of NOD mice versus C57BL/6 mice. Control equals nonsensitized mice. HEL, HEL-sensitized mice; HEL+D3, HEL-sensitized and 1,25(OH)2D3-treated mice. *P < 0.05. Each bar is the mean of n = 8–15 mice (±SE).s', 百拇医药
Finally, NOD-scid mice lacking T- and B-cells were primed and rechallenged with HEL/CFA. Interestingly, in these mice (n = 6), no symptoms of shock were observed, conclusively demonstrating the key role of T-cells in the observed phenomenon.s', 百拇医药
Involvement of platelet activation in shock.s', 百拇医药
Because the clinical picture of lethal shock was accompanied by hypercoagulation, compatible with anaphylaxis leading to death by disseminated intravasal coagulation, we questioned the involvement of platelet activation in induced shock. We investigated if factors involved in this pathway showed an abnormal expression profile in the NOD mouse or were influenced by 1{alpha} ,25(OH)2D3. Therefore, mRNA levels for PAF-R and PAF-AH, the enzyme most responsible for degradation of PAF, were quantified by real-time RT-PCR in the spleens of NOD and C57BL/6 mice 3 weeks after the first footpad injection with HEL.
Interestingly, a clear inter-strain difference in the expression of PAF-R mRNA was observed, with 2.5-fold higher levels in NOD mice than in C57BL/6 mice (P < 0.05, ). Additionally, 1{alpha} ,25(OH)2D3 administration further increased the expression PAF-R mRNA in NOD as well as C57BL/6 mice by 1.5- and 2.5-fold, respectively (P < 0.05, ). These results, showing a regulation of the PAF-R in the analyzed system, are highly indicative for an important role of the PAF pathway in induction and sensitivity for anaphylaxis.2qw, 百拇医药
fig.ommtted2qw, 百拇医药
Impact of HEL immunization on PAF-R (A) and PAF-AH (B) mRNA levels in spleens of NOD or C57BL/6 mice not treated (HEL) or treated with 1,25(OH)2D3 (HEL+D3). *P < 0.05; **P < 0.01. Each bar is the mean of n = 8–15 mice (±SE).2qw, 百拇医药
The mRNA expression of PAF-AH in the spleens of HEL-immunized NOD mice was 1.5-fold higher than that in C57BL/6 mice (P < 0.01). 1{alpha} ,25(OH)2D3 treatment did not influence the PAF-AH mRNA levels. A minor interstrain difference remained after 125(OH)2D3 treatment; however, this difference did not reach significance
To conclusively demonstrate the involvement of the platelet activation pathway in the shock phenomenon, we attempted to rescue the HEL-immunized NOD mice from lethal shock by administration of a PAF-R antagonist. Indeed, mortality was completely abrogated by administration of a single dose of 500 µg of the PAF-R antagonist WEB2086 10 min before the HEL booster in all HEL-immunized NOD mice, irrespective of 1{alpha} ,25(OH)2D3 treatment . Only mild nonlethal shock symptoms were observed in 75% of the rescued mice; in each group, shock was completely prevented in two of eight mice, whereas the other six developed milder shock symptoms, from which they all recovered .g, http://www.100md.com
fig.ommttedg, http://www.100md.com
Effect of rescue treatment with WEB2086 on shock incidence and severity in NOD mice. HEL, HEL-sensitized; HEL+D3, HEL-sensitized, 1,25(OH)2D3-treated; HEL+D3+WEB, HEL-sensitized, 1,25(OH)2D3-treated, rescued with WEB2086; HEL+WEB, HEL-sensitized, rescued with WEB2086. No signs of shock; reversible shock; , lethal shock. *P < 0.05 concerning disease incidence. n = 8 mice per group.
DISCUSSION+'3, 百拇医药
Major advances have recently been made in better understanding the pathogenesis of various autoimmune diseases. Data obtained from animal models of human type 1 diabetes and MS suggest that, in these diseases, a pivotal role in immune self-destruction is played by Th1 cells (17). Efforts have thus been made to find ways of promoting T-helper differentiation toward a Th2 rather than Th1 subtype, thereby arresting the autoimmune destructive process (5). It has been observed that a protective switch of T-helper differentiation could be obtained by vaccination with disease-triggering autoantigens (18,19). Moreover, this strategy has been extrapolated to human MS (10) and is about to be adopted in human type 1 diabetes (8,20). However, initial enthusiasm was tempered by the observation that vaccination with self-antigens could elicit anaphylactic shock in the context of a disturbed immune system (11,12). This anaphylactic reaction could be triggered by exactly the same T-cell differentiation shift that provided protection against autoimmunity (21). In contrast to a Th1-mediated autoimmune attack, allergic disease is mainly Th2 dependent (22) and is largely mediated by IgE (22,23). Aggressive allergic reactions to encephalitogenic antigens were also observed when the vaccination approach was tested in human MS (12,24). The benefit of protection against an autoimmune disease could therefore be hampered by the allergic side effects.
NOD mice are animals that have a special immune system, with particularities of both T-cells and antigen-presenting cells (2). Although their most known immune feature is the Th1-driven destruction of pancreatic islets (4), literature data about their Th2 activity are conflicting (25,26). The initial aim of our study was to check the safety of antigenic vaccination in this particular strain with respect to anaphylaxis. The set of antigens tested included diabetes-relevant autoantigens (GAD65, mouse insulin, and hsp65) and diabetes-irrelevant self (PLP) or nonself (HEL, OVA, KLH, and TT) antigens. The behavior of NOD mice to vaccination was compared with that of two other mouse strains: C57BL/6 and BALB/c mice. To force T-cell differentiation toward a Th2 setting, we submitted groups of mice from the three strains to therapy with 1{alpha} ,25(OH)2D3. The active form of vitamin D is known as a powerful modulator of the immune system (27). The 1{alpha} ,25(OH)2D3-induced shift toward Th2 differentiation can even prevent the onset of Th1-mediated autoimmune diseases, such as diabetes in NOD mice (28) or EAE in SJL/J mice (29).
The only antigen that provoked anaphylactic reactions in our experiments was HEL. However, mice from various strains behaved in different ways. C57BL/6 mice were completely resistant to HEL-induced anaphylaxis, irrespective of 1{alpha} ,25(OH)2D3 administration. This outcome was not unexpected because C57BL/6 mice are known for having a predominantly Th1-driven immune response, though not accompanied by autoimmune phenomena (30). Although T-cell lymphocyte differentiation is mostly Th2-directed in BALB/c mice (30), HEL vaccination by itself did not evolve to shock. When the immune system was further stressed toward Th2 responses by 1{alpha} ,25(OH)2D3 administration, all BALB/c mice nevertheless developed a mild reversible form of shock. Unexpectedly, NOD mice, known for their Th1-mediated immune reactivity, also developed shock consecutive to HEL sensitization. Moreover, shock appeared in NOD mice even without any modulation of the immune system with 1{alpha} ,25(OH)2D3 and was rather aggressive (even lethal for certain animals). 1{alpha} ,25(OH)2D3 administration further increased mortality in HEL-sensitized NOD mice.
We were surprised by the strain-selective anaphylactic shock induced by HEL in NOD mice. This mouse strain is indeed known to react through Th1-mediated mechanisms (2–4,25) and was moreover described as having a defect in antigen presentation (31,32). An exacerbation of anaphylaxis caused by the administration of 1{alpha} ,25(OH)2D3 was also not immediately clear because previous studies suggested that a 1{alpha} ,25(OH)2D3-dependent differentiation shift toward Th2 takes place exclusively in the presence of diabetes-relevant autoantigens (33). We decided therefore to search for the mechanisms underlying the strain-specific behavior of the NOD immune system.4k/, 百拇医药
Complete rescue by therapy with dexamethasone demonstrated undoubtedly that HEL-induced shock in NOD mice depended on immune effectors. Furthermore, the complete absence of symptoms in NOD-scid mice lacking T- and B-cells (34) proved the indispensable role of T-cells in HEL-induced shock. Cytokine modifications caused by 1{alpha} ,25(OH)2D3 in NOD mice were partially suggestive for a Th1 to Th2 shift, with a decrease in Th1-secreted IFN-{gamma} and a concomitant increase in Th2-specific IL-13. However, no differences in IL-12 and IL-4 levels were found between the 1{alpha} ,25(OH)2D3-treated and nontreated HEL-sensitized NOD mice. Thus, these changes did not completely overlap the 1{alpha} ,25(OH)2D3-dependent modifications found when the immune system was activated by autoantigens (33), suggesting antigen-specific immune modulation by 1{alpha} ,25(OH)2D3. Comparison of cytokine mRNA spectra between HEL-immunized shock-sensitive NOD mice and shock-resistant C57BL/6 mice showed differences in IL-13 mRNA levels, which were further accentuated by 1{alpha} ,25(OH)2D3 administration. Therefore, our data supported that the cytokine has a critical role in the particular behavior of NOD mice toward HEL sensitization. IL-13 is a Th2-specific cytokine closely related to IL-4 (35) and synthesized in both humans and mice (36). IL-13 has been alleged to be importantly involved in Th2-initiated allergic reactions, including anaphylaxis (37). The significant difference in serum IgE levels between the two mouse strains provided evidence of IgE-mediated anaphylactic shock in NOD mice. IgE is an immunoglobulin involved in most allergic reactions (13,38). The parallelism between IL-13 mRNA levels and serum IgE levels assessed in our mice stresses a causal relationship between the two immune parameters. Direct stimulation of IgE synthesis by IL-13 has been actually described (38). Certain NOD mice also displayed higher levels of Th2-dependent IgG1. IgG1 may by itself induce anaphylactic shock in mice (11,13).
PAF is an important mediator of immune, hemostatic, and allergic reactions (39). This bioactive phospholipid can be regarded as a cytokine because of its multiple actions on a wide range of cells (including neutrophils, monocytes, and platelets) and tissues that possess specific membrane receptors (PAF-R) (40). The acute inflammatory effects of PAF are largely due to the stimulation of neutrophilic granulocytes as the first line of defense (40). Exaggerated synthesis of PAF or defective mechanisms of PAF inactivation can however lead to pathologic inflammation, shock, and thrombosis (39). There are data in the literature suggesting significant interference of 1,25(OH)2D3 with PAF actions (41) or metabolism (42). Because 1,25(OH)2D3 had such a powerful impact on shock incidence and severity, we suspected an interference of PAF in HEL-induced shock. PAF-R mRNA levels were significantly higher in NOD mice, pleading in favor of an increased sensitivity to PAF inflammatory actions in this strain. In concordance with other studies (43), 1,25(OH)2D3 therapy caused a further increase in the PAF-R expression, a phenomenon that might partly explain the increase in shock severity. Surprisingly, PAF-AH mRNA levels in HEL-sensitized NOD mice were significantly higher than those in C57BL/6 mice. PAF-AH is the main enzyme involved in PAF degradation. The increase in PAF-AH mRNA levels after HEL sensitization could be the expression of a natural feedback reaction against exaggerated PAF synthesis (44). Contrary to another study (42), 1,25(OH)2D3 therapy did not significantly inhibit PAF-AH mRNA expression in HEL-sensitized NOD mice. However, the minor interstrain difference of PAF-AH levels was no longer significant in the 1,25(OH)2D3-treated groups. The direct proof of PAF involvement in the etiopathogenesis of strain-specific HEL-induced shock was obtained when rescue therapy with a PAF-R antagonist (WEB2086) showed to be efficient in preventing shock or diminishing its severity. The activation of the Th2 arm of the immune attack is probably linked to the activation of the PAF system. IgE, for example, can undeniably cause direct stimulation of PAF synthesis by various cells, such as mastocytes (41). Moreover, a recent article (45) suggested that murine antigen-induced anaphylaxis is primarily mediated by the PAF pathway and not by the classic IgE pathway.
Finally the intriguing question remains why HEL induces anaphylaxis in the NOD mouse model, as opposed to the other antigens tested. An extensive GenBank search did not reveal any peptide homology between HEL and any of the known diabetes-relevant autoantigens, not excluding, however, a possible similarity with an as yet undefined autoantigen.5iawd, 百拇医药
In conclusion, our data clearly demonstrate the appearance of a severe Th2-biased immune reaction after challenge with a widely used control antigen in a mouse strain known mainly for its genetic propensity toward Th1-mediated autoimmune destruction of ß-cells. Strain specificity of this reaction points out that immune systems with a disturbed genetic background may reply unpredictably to an immune challenge, even with "innocent" antigens. Autoimmunity should therefore not be simply viewed as an isolated particularity of the immune response but rather as a general disturbance of the immune system. It is currently accepted that Th1-oriented autoimmunity is involved in the pathogenesis of human type 1 diabetes (1–5). Shifting the activation of the immune system toward the Th2 differentiation pathway has been proven efficient in preventing diabetes in animal models (2,3,5) and might be regarded as an interesting alternative to type 1 diabetes prevention in humans (5,9). The immune system of type 1 diabetic patients may, however, manifest changes beyond the local islet-destructive effect (46). Our data complete the conflicting experience already existing with vaccination strategies in EAE and human MS (11,21,24). Extreme caution is therefore needed when adopting immune-modulating strategies, taking into consideration that a shift in T-helper differentiation might always represent a transfer of risk from a Th1-mediated type IV delayed immune attack to a Th2-mediated anaphylactic reaction, especially in situations with an already-activated Th2 system, such as a coincidental infection or allergy.
ACKNOWLEDGMENTS$z|8, 百拇医药
This work was supported by the Flemish Research Foundation (Fonds voor Wetenschappelijk Onderzoek [FWO] grant G.0084.02), the Interuniversity Attraction Poles (IUAP/P5/03/20), the European Foundation for the Study of Diabetes, the Flemish Governments of Research and Education (Geconerteerde Onderzoeksactic [GOA] 99/10), and an FWO fellowship for Chantal Mathieu.$z|8, 百拇医药
The expert technical assistance of Jos Laureys is gratefully acknowledged.$z|8, 百拇医药
REFERENCES$z|8, 百拇医药
Yoon JW, Jun HS: Cellular and molecular pathogenic mechanisms of insulin-dependent diabetes mellitus. Ann N Y Acad Sci928 :200 –211,2001$z|8, 百拇医药
Adorini L, Gregori S, Harrison LC: Understanding autoimmune diabetes: insights from mouse models. Trends Mol Med8 :31 –38,2002$z|8, 百拇医药
Rabinovitch A: An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes Metab Rev14 :129 –151,1998$z|8, 百拇医药
Wong FS, Janeway CA Jr: Insulin-dependent diabetes mellitus and its animal models. Curr Opin Immunol11 :643 –647,1999
Casares S, Brumeanu TD: Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies. Curr Mol Med1 :357 –378,2001-e7&(, 百拇医药
Kundig TM, Bachmann MF, Ohashi PS, Pircher H, Hengartner H, Zinkernagel RM: On T cell memory: arguments for antigen dependence. Immunol Rev150 :63 –90,1996-e7&(, 百拇医药
Foucras G, Gallard A, Coureau C, Kanellopoulos JM, Guery JC: Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with Th2 phenotype acquisition in vivo. J Immunol168 :179 –187,2002-e7&(, 百拇医药
Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen IR: ß-Cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind phase II trial. Lancet358 :1749 –1753,2002-e7&(, 百拇医药
Ramiya VK, Lan MS, Wasserfall CH, Notkins AL, Maclaren NK: Immunization therapies in the prevention of diabetes. J Autoimmun10 :287 –292,1997-e7&(, 百拇医药
Willenborg DO, Staykova MA: Approaches to the treatment of central nervous system autoimmune disease using specific neuroantigen. Immunol Cell Biol76 :91 –103,1998
Pedotti R, Mitchell D, Wedemeyer J, Karpuj M, Chabas D, Hattab EM, Tsai M, Galli SJ, Steinman L: An unexpected version of horror autotoxicus: anaphylactic shock to a self-peptide. Nat Immunol2 :216 –222,20010|pjki, 百拇医药
Kappos L, Comi G, Panitch H, Oger J, Antel J, Conlon P, Steinman L, The Altered Peptide Ligand in Relapsing MS Study Group: Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. Nat Med6 :1176 –1182,20000|pjki, 百拇医药
Harada M, Nagata M, Takeuchi M, Makino S, Aburahi S: High susceptibility of cataract Shionogi (CTS) mice to passive anaphylactic shock mediated by allogeneic IgE and IgG1 monoclonal antibodies. Immunol Invest20 :305 –315,19910|pjki, 百拇医药
Hurtenbach U, Lier E, Adorini L, Nagy ZA: Prevention of autoimmune diabetes in non-obese diabetic mice by treatment with a class II major histocompatibility compex-blocking peptide. J Exp Med177 :1499 –1504,19930|pjki, 百拇医药
Pozilli P, Signore A, Williams AJ, Beales P: NOD mouse colonies around the world: recent facts and figures. Immunol Today14 :193 –196,1993
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods25 :386 –401,2001n&, http://www.100md.com
Singh VK, Mehrotra S, Agarwal SS: The paradigm of Th1 and Th2 cytokines: its relevance to autoimmunity and allergy. Immunol Res20 :147 –161,1999n&, http://www.100md.com
Parish NM, Hutchings PR, O’Reilly L, Quartey-Papafio R, Healey D, Ozegbe P, Cooke A: Tolerance induction as a therapeutic strategy for the control of autoimmune endocrine disease in mouse models. Immunol Rev144 :269 –300,1995n&, http://www.100md.com
Bach JF, Chatenoud L: Tolerance to islet autoantigens in type 1 diabetes. Annu Rev Immunol19 :131 –161,2001n&, http://www.100md.com
Simone EA, Wegmann DR, Eisenbarth GS: Immunologic "vaccination" for the prevention of autoimmune diabetes (type 1A). Diabetes Care22 (Suppl. 2) :B7 –B15,1999n&, http://www.100md.com
Silverstein AM: Autoimmunity versus horror autotoxicus: the struggle for recognition. Nat Immunol2 :279 –281,2001n&, http://www.100md.com
Broide DH: Molecular and cellular mechanisms of allergic disease. J Allergy Clin Immunol108 :S65 –S71,2001
Kay AB: Allergy and allergic diseases: first of two parts. N Engl J Med344 :30 –37,2001jmd^, http://www.100md.com
Wiendl H, Neuhaus O, Kappos L, Hohlfeld R: Multiple sclerosis: current view of failed and discontinued clinical trials of drug treatment. Nervenarzt71 :597 –610,2000jmd^, http://www.100md.com
Hartemann AH, Richard MF, Boitard C: Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice. Clin Exp Immunol116 :225 –230,1999jmd^, http://www.100md.com
Schloot NC, Hanifi-Moghaddam P, Goebel C, Shatavi SV, Flohe S, Kolb H, Rothe H: Serum IFN-gamma and IL-10: levels are associated with disease progression in non-obese diabetic mice. Diabetes Metab Res Rev18 :64 –70,2002jmd^, http://www.100md.com
Mathieu C, Adorini L: The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents. Trends Mol Me8 :174 –179,2002jmd^, http://www.100md.com
Mathieu C, Laureys J, Sobis H, Vandeputte M, Waer M, Bouillon R: 1,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice. Diabetes41 :1491 –1495,1992jmd^, http://www.100md.com
Branisteanu DD, Waer M, Sobis H, Marcelis S, Vandeputte M, Bouillon R: Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 alpha, 25-(OH)2D3. J Neuroimmunol6 :151 –160,1995
Hayashi T, Hasegawa K, Nakai S, Hamachi T, Adachi Y, Yamauchi Y, Maeda K: Bronchial lesions of the late asthmatic response in Balb/c and C57BL/6 mice. J Comp Pathol125 :208 –213,2001re@qgv, 百拇医药
Strid J, Lopes L, Marcinkiewicz J, Petrovska L, Nowak B, Chain BM, Lund T: A defect in bone marrow derived dendritic cell maturation in the nonobese diabetic mouse. Clin Exp Immunol123 :375 –381,2001re@qgv, 百拇医药
Piganelli JD, Martin T, Haskins K: Splenic macrophages from the NOD mouse are defective in the ability to present antigen. Diabetes47 :1212 –1218,1998re@qgv, 百拇医药
Overbergh L, Decallonne B, Waer M, Rutgeerts O, Valckx D, Casteels KM, Laureys J, Bouillon R, Mathieu C: 1alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524–543). Diabetes49 :1301 –1307,2000re@qgv, 百拇医药
Shultz L, Schweitzer P, Christianson S, Gott B, Schweitzer I, Tennent B, McKenna S, Mobraaten L, Rajan T, Greiner D, Leiter E: Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol154 :180 –191,1995
Zurawski G, de Vries JE: Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today15 :19 –26,199488%, 百拇医药
Brombacher F: The role of interleukin-13 in infectious diseases and allergy. Bioessays22 :646 –656,200088%, 百拇医药
Fallon PG, Emson CL, Smith P, McKenzie AN: IL-13 overexpression predisposes to anaphylaxis following antigen sensitization. J Immunol166 :2712 –2716,200188%, 百拇医药
Corry DB, Kheradmand F: Induction and regulation of the IgE response (Review Article) Nature402 (Suppl. 6760) :B18 –B23,199988%, 百拇医药
Ayala A, Chaudry IH: Platelet activating factor and its role in trauma, shock, and sepsis. New Horiz4 :265 –275,199688%, 百拇医药
Snyder F: Platelet-activating factor and its analogs: metabolic pathways and related intracellular processes. Biochim Biophys Acta1254 :231 –249,199588%, 百拇医药
Chen SF, Ruan YJ: 1 alpha,25-Dihydroxyvitamin D3 decreases scalding- and platelet-activating factor-induced high vascular permeability and tissue oedema. Pharmacol Toxicol76 :365 –367,199588%, 百拇医药
Narahara H, Miyakawa I, Johnston JM: The inhibitory effect of 1,25-dihydroxyvitamin D3 on the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages. J Clin Endocrinol Metab80 :3121 –3126,199588%, 百拇医药
Muller E, Dupuis G, Turcotte S, Rola-Pleszczynski M: Human PAF receptor gene expression: induction during HL-60 cell differentiation. Biochem Biophys Res Commun181 :1580 –1586,199188%, 百拇医药
Stafforini DM, McIntyre TM, Zimmerman GA, Prescott SM: Platelet-activating factor acetylhydrolases. J Biol Chem272 :17895 –17898,199788%, 百拇医药
Strait RT, Morris SC, Yang M, Qu XW, Finkelman FD: Pathways of anaphylaxis in the mouse. J Allergy Clin Immunol109 :658 –668,200288%, 百拇医药
Friday RP, Trucco M, Pietropaolo M: Genetics of type 1 diabetes mellitus. Diabetes Nutr Metab12 :3 –26,1999(Lut Overbergh Brigitte DecallonneDumitru D. Branisteanu Dirk Valck Ahmad Kasran Roger Bouillon and C)